SRP193951 PRJNA539885 Spacer acquisition from RNA mediated by a natural reverse transcriptase-Cas1 fusion protein associated to a type III-D CRISPR-Cas System in Vibrio vulnificus Association of reverse transcriptases (RTs) to CRISPR-Cas system has recently taken attention because the RT activity appears to facilitate the RT-dependent acquisition of spacers from RNA molecules. In spite of the great phylogenetically diversity of RTs associated to CRISPR-Cas systems, current knowledge on this type of spacer acquisition process offers limited insight. Here, we characterize the in vivo acquisition of spacers mediated by a RT-Cas1 fusion protein belonging to clade 6 linked to a type III-D system from Vibrio vulnificus strain YJ016 and show that the adaptive module consisting of the RT-Cas1 fusion, two distinct Cas2 proteins (A and B) and one of the two CRISPR arrays are completely functional in a heterologous host (E. coli). We found that mutation in the active site of the RT domain significantly decreased the acquisition of new spacers and show that this RT-Cas1 associated adaptation module is able to incorporate into the CRISPR array spacers from RNA molecules. We demonstrate that the two different Cas2 protein of the adaptation module, present in all members of this clade are required for spacer acquisition. Furthermore, we found that there are several sequence specific requirements for the correct acquisition and integration of spacers that can be acquired from any region of the genome with no bias along the 5´and 3´end of coding sequences, which could make this RT-Cas1 system a good alternative for biotechnological applications. Our study provides novel insights into the acquisition of spacer from RNA molecules mediated by RT-Cas1 fusion proteins. Webpage of the group https://www.eez.csic.es/en/structure-dynamics-and-function-rhizobacterial-genomes